Exogenous DNA Damage Research Articles

Between Life and Death: Sea Urchin Embryos Undergo Peculiar DNA Fragmentation after Exposure to Vanadium, Cadmium, Gadolinium, and Selenium.

Exogenous DNA damage represents one of the most harmful outcomes produced by environmental, physical, or chemical agents. Here, a comparative analysis of DNA fragmentation was carried out on Paracentrotus lividus sea urchin embryos exposed to four common pollutants of the marine environment: vanadium, cadmium, gadolinium and selenium. Using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, fragmented DNA was quantified and localized in apoptotic cells mapping whole-mount embryos. This is the first study reporting how different chemicals are able to activate distinctive apoptotic features in sea urchin embryos, categorized as follows: (i) cell-selective apoptosis, showing DNA fragmentation restricted to a subset of extremely damaged cells, acting as an embryo survival mechanism; or (ii) total apoptosis, with fragmented DNA widespread throughout the cells of the entire embryo, leading to its death. Also, this is the first report of the effects of Se exposure on P. lividus sea urchin embryos. These data confirm the TUNEL assay as the most suitable test to study DNA fragmentation in the sea urchin embryo model system. Taken together, this research highlights embryos' ability to find alternative pathways and set physiological limits for development under stress conditions.

Dimethyl Sulfoxide Attenuates Ionizing Radiation-induced Centrosome Overduplication and Multipolar Cell Division in Human Induced Pluripotent Stem Cells.

Centrosomes are important organelles for cell division and genome stability. Ionizing radiation exposure efficiently induces centrosome overduplication via the disconnection of the cell and centrosome duplication cycles. Over duplicated centrosomes cause mitotic catastrophe or chromosome aberrations, leading to cell death or tumorigenesis. Pluripotent stem cells, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), can differentiate into all organs. To maintain pluripotency, PSCs show specific cellular dynamics, such as a short G1 phase and silenced cell-cycle checkpoints for high cellular proliferation. However, how exogenous DNA damage affects cell cycle-dependent centrosome number regulation in PSCs remains unknown. This study used human iPSCs (hiPSCs) derived from primary skin fibroblasts as a PSC model to address this question. hiPSCs derived from somatic cells could be a useful tool for addressing the radiation response in cell lineage differentiation. After radiation exposure, the hiPSCs showed a higher frequency of centrosome overduplication and multipolar cell division than the differentiated cells. To suppress the indirect effect of radiation exposure, we used the radical scavenger dimethyl sulfoxide (DMSO). Combined treatment with radiation and DMSO efficiently suppressed DNA damage and centrosome overduplication in hiPSCs. Our results will contribute to the understanding of the dynamics of stem cells and the assessment of the risk of genome instability for regenerative medicine.

NuRD chromatin remodeling is required to repair exogenous DSBs in the Caenorhabditis elegans germline.

Organisms rely on coordinated networks of DNA repair pathways to protect genomes against toxic double-strand breaks (DSBs), particularly in germ cells. All repair mechanisms must successfully negotiate the local chromatin environment in order to access DNA. For example, nucleosomes can be repositioned by the highly conserved Nucleosome Remodeling and Deacetylase (NuRD) complex. In Caenorhabditis elegans, NuRD functions in the germline to repair DSBs - the loss of NuRD's ATPase subunit, LET-418/CHD4, prevents DSB resolution and therefore reduces fertility. In this study, we challenge germlines with exogenous DNA damage to better understand NuRD's role in repairing DSBs. We find that let-418 mutants are hypersensitive to cisplatin and hydroxyurea: exposure to either mutagen impedes DSB repair, generates aneuploid oocytes, and severely reduces fertility and embryonic survival. These defects resemble those seen when the Fanconi anemia (FA) DNA repair pathway is compromised, and we find that LET-418's activity is epistatic to that of the FA component FCD-2/FANCD2. We propose a model in which NuRD is recruited to the site of DNA lesions to remodel chromatin and allow access for FA pathway components. Together, these results implicate NuRD in the repair of both endogenous DSBs and exogenous DNA lesions to preserve genome integrity in developing germ cells.

Abstract 5170: Novel dSTRIDE-HR assays can accurately assess DNA repair defects in human cancers and distinguish HR status of tumors

Abstract Accurate patient stratification is crucial for the successful application of targeted anti-cancer therapeutic approaches. It has been shown that PARP inhibitors are synthetic lethal with defective homologous recombination (HR) repair, with genes such as BRCA1 and BRCA2 identified as conferring therapy resistance or sensitivity, depending on their mutational status. Moreover, there is evidence of patient benefit of carboplatin-based therapy in HR deficient (HRD) tumors. With a growing emphasis on HRD in cancer treatment, efforts have been made to define the HRD phenotype. Nonetheless, the current approaches employed in the clinic, such as Next Generation Sequencing (NGS), HRD score or RAD51 assays have significant limitations and their interpretation can be unclear. To answer the need for a functional HR-specific biomarker, we have recently developed and introduced two new assays, called collectively dSTRIDE-HR. The assays are based on the STRIDE platform technology that enables direct and sensitive detection of single- or double-strand DNA breaks in situ in fixed cells. Novel dSTRIDE-HR explicitly detect double-strand DNA breaks localized in close proximity to RPA (dSTRIDE-RPA) or RAD51 (dSTRIDE-RAD51) proteins. The assays have been shown to be reliable reporters of HR activation in cellular models after exogenous DNA damage induction, yet their potential to predict HR status in an in vivo setting has not been tested so far. To assess the suitability of the dSTRIDE-HR assays for determining the HR status of patients’ tumor cells, we applied these assays in FFPE sections from biopsies derived from breast cancer patients with a different mutational status of BRCA1 and BRCA2 genes. The assays showed low dSTRIDE-RPA and dSTRIDE-RAD51 readouts in BRCA1/2 mutated tumors in comparison to wild type tumors, as well as a great concordance of readouts within a given genetic background. Additionally, the cell cycle analysis proved nearly twofold increase in foci numbers of dSTRIDE-RPA and dSTRIDE-RAD51 in G2/S phase. Our study unveils a potential of dSTRIDE-HR assays to become predictive biomarkers reporting on the status of HR repair in breast tumors, although more patient samples need to be tested to confirm initial findings. Citation Format: Monika Jarosz, Zsombor Prucsi, Franek Sierpowski, Karolina Stepien, Sophie Ostrowska-Paton, Szymon Koman, Nadeem Riaz, Simon Powell, Fresia Pareja, Edaise da Silva, Magdalena Kordon-Kiszala, Kamil Solarczyk. Novel dSTRIDE-HR assays can accurately assess DNA repair defects in human cancers and distinguish HR status of tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5170.

Abstract 591: Discovery of ZM-2322, a highly selective, potent inhibitor of membrane-associated tyrosine- and threonine-specific cdc2-inhibitory kinase (PKMYT1)

Abstract The genome DNA is consistently threatened by a variety of intrinsic and exogenous DNA damage factors. Cell cycle checkpoint is one important mechanism in response to DNA damage and maintaining genome stability. In tumor cells, gene alterations, such as CCNE1 amplification (encoding for Cyclin E1), result in G1/S checkpoint loss and replication stress, leading to high dependency on the G2/M checkpoint. PKMYT1 inhibits crucial checkpoint protein CDK1 activity by phosphorylating CDK1 at the residues of Thr-14 and Tyr-15 in the G2/M phase. In CCNE1 amplification cells, the inhibition of PKMYT1 leads to the reduction of pCDK1 and loss of G2/M checkpoint, consequently results in premature mitosis and the accumulation of DNA damage, then finally induces cell death. Here we identify a novel small molecular ZM-2322, which displays robust intracellular binding to PKMYT1 and inhibits the specific downstream phosphorylation of CDK1 Thr-14 with IC50 values of 14 nM and 29 nM, respectively. ZM-2322 strongly inhibits the proliferation of the HCC1569 cell line with CCNE1 high-level amplification and shows a >400 × selectivity folds over a CCNE1 wildtype cell line SKOV3. ZM-2322 also shows significant inhibition of tumor growth in a dose-dependent manner in the HCC1569 xenograft model, and the efficacy correlates well with PK and target inhibition. There is evidence showing that loss-of-function mutations of FBXW7, a cyclin E E3 ligase, lead to cyclin E accumulation and are synthetic-lethal with PKMYT1 inhibition. Consistently, a potent anti-proliferation of ZM-2322 is observed in DLD-1 FBXW7 -/- cells, but not DLD-1 parental cells. These data suggest a high potency of ZM-2322 on PKMYT1 inhibition and a potential good kinase selectivity which is further confirmed by using a kinase panel biochemical assay. As expected, when compared head-to-head with another clinical PKMYT1 inhibitor RP-6306 which shows inhibitory effect on 27 of 217 human kinases with inhibition rates >90% at 1 μM, ZM-2322 is found to be a much more PKMYT1-selective inhibitor and hits only several individual kinases under the same assay condition, suggesting a potentiality in better safety profile and higher dose tolerance. Furthermore, our data illustrates that in combination with gemcitabine, ZM-2322 elicits strong synergetic anti-proliferation activities on HCC1569 cells in vitro and results in tumor regression with a minor effect on body weight in vivo. Besides, a strong synergy effect is also observed in the combo of ZM-2322 and ATR inhibitor in HCC1569 in vitro. These data indicate that ZM-2322 has the potential of application expansion and good tolerance under continuous administration. Taken together, our data suggest that ZM-2322 is a potent and highly selective PKMYT1 inhibitor. Citation Format: Feng Zhou, Lu Liu, Lei Jiang, Baoying Cheng, Zhentao Li, Dongxing Zhu, Jianbin Xue, Liting Xue, Renhong Tang. Discovery of ZM-2322, a highly selective, potent inhibitor of membrane-associated tyrosine- and threonine-specific cdc2-inhibitory kinase (PKMYT1) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 591.

Characteristics of germline DNA damage response gene mutations in ovarian cancer in Southwest China

DNA damage response (DDR) pathways are responsible for repairing endogenous or exogenous DNA damage to maintain the stability of the cellular genome, including homologous recombination repair (HRR) pathway, mismatch repair (MMR) pathway, etc. In ovarian cancer, current studies are focused on HRR genes, especially BRCA1/2, and the results show regional and population differences. To characterize germline mutations in DDR genes in ovarian cancer in Southwest China, 432 unselected ovarian cancer patients underwent multi-gene panel testing from October 2016 to October 2020. Overall, deleterious germline mutations in DDR genes were detected in 346 patients (80.1%), and in BRCA1/2 were detected in 126 patients (29.2%). The prevalence of deleterious germline mutations in BRCA2 is higher than in other studies (patients are mainly from Eastern China), and so is the mismatch repair genes. We identified three novel BRCA1/2 mutations, two of which probably deleterious (BRCA1 p.K1622* and BRCA2 p.L2987P). Furthermore, we pointed out that deleterious mutations of FNACD2 and RECQL4 are potential ovarian cancer susceptibility genes and may predispose carriers to ovarian cancer. In conclusion, our study highlights the necessity of comprehensive germline mutation detection of DNA damage response genes in ovarian cancer patients, which is conducive to patient management and genetic counseling.

FLIP(C1orf112)-FIGNL1 complex regulates RAD51 chromatin association to promote viability after replication stress

Homologous recombination (HR) plays critical roles in repairing lesions that arise during DNA replication and is thus essential for viability. RAD51 plays important roles during replication and HR, however, how RAD51 is regulated downstream of nucleofilament formation and how the varied RAD51 functions are regulated is not clear. We have investigated the protein c1orf112/FLIP that previously scored in genome-wide screens for mediators of DNA inter-strand crosslink (ICL) repair. Upon ICL agent exposure, FLIP loss leads to marked cell death, elevated chromosomal instability, increased micronuclei formation, altered cell cycle progression and increased DNA damage signaling. FLIP is recruited to damage foci and forms a complex with FIGNL1. Both proteins have epistatic roles in ICL repair, forming a stable complex. Mechanistically, FLIP loss leads to increased RAD51 amounts and foci on chromatin both with or without exogenous DNA damage, defective replication fork progression and reduced HR competency. We posit that FLIP is essential for limiting RAD51 levels on chromatin in the absence of damage and for RAD51 dissociation from nucleofilaments to properly complete HR. Failure to do so leads to replication slowing and inability to complete repair.

Abstract A033: The ING5 epigenetic reader regulates a subset of DNA repair genes to maintain genomic integrity

Abstract The ING family of epigenetic histone code readers (ING1-5) recognize the H3K4Me3 epigenetic mark to target histone acetyltransferase (HAT) or histone deacetylase (HDAC) complexes to regulate local acetylation levels and affect transcription. We and others have shown that ING5 promotes stem cell character in normal and in cancer stem cells and that loss of ING5 promotes stem cell differentiation. Examination of an ING5 knockout (KO) mouse model showed increased levels of DNA damage in different tissues, and particularly in the male germ line. ING5 mouse embryo fibroblasts (MEFs) also show a >2-fold increase in cells containing gH2AX foci in the absence of exogenous DNA damaging agents. Examining the transcriptomes of ING5 KO and wild-type littermate MEFs indicates that several genes encoding key DNA repair proteins such as those acting in homologous recombination (BRCA1 and BRCA2) and base excision repair (PARP1) are repressed several-fold and ING5 KO MEFs show increased sensitivity to the PARP inhibitor olaparib. Given that ING5 functions to help maintain stem cell character in normal and in cancer stem cells, we propose that ING5 serves a role in maintaining a high level of DNA repair capability in stem cell populations to preserve genomic integrity. Citation Format: Arthur E Dantas, Buthaina Al-Shueili, Mahbod Djamshidi, Karl Riabowol. The ING5 epigenetic reader regulates a subset of DNA repair genes to maintain genomic integrity [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A033.

P53 and p21 dynamics encode single-cell DNA damage levels, fine-tuning proliferation and shaping population heterogeneity

Cells must accurately and quickly detect DNA damage through a set of checkpoint mechanisms that enable repair and control proliferation. Heterogeneous levels of cellular stress and noisy signaling processes can lead to phenotypic variability but little is known about their role in underlying proliferation heterogeneity. Here we study two previously published single cell datasets and find that cells encode heterogeneous levels of endogenous and exogenous DNA damage to shape proliferation heterogeneity at the population level. Using a comprehensive time series analysis of short- and long-term signaling dynamics of p53 and p21, we show that DNA damage levels are quantitatively translated into p53 and p21 signal parameters in a gradual manner. Analyzing instantaneous proliferation and signaling differences among equally-radiated cells, we identify time-localized changes in the period of p53 pulses that drive cells out of a low proliferative state. Our findings suggest a novel role of the p53-p21 network in quantitatively encoding DNA damage strength and fine-tuning proliferation trajectories.

Xrcc5/KU80 is not required for the survival or activation of prophase-arrested oocytes in primordial follicles.

The non-growing, meiotically-arrested oocytes housed within primordial follicles are exquisitely sensitive to genotoxic insults from endogenous and exogenous sources. Even a single DNA double-strand break (DSB) can trigger oocyte apoptosis, which can lead to accelerated depletion of the ovarian reserve, early loss of fertility and menopause. Therefore, repair of DNA damage is important for preserving the quality of oocytes to sustain fertility across the reproductive lifespan. This study aimed to evaluate the role of KU80 (encoded by the XRCC5 gene) - an essential component of the non-homologous end joining (NHEJ) pathway - in the repair of oocyte DNA DSBs during reproductive ageing, and following insult caused by the DNA-damaging chemotherapies cyclophosphamide and cisplatin. To investigate the importance of KU80 following endogenous and exogenous DNA damage, ovaries from conditional oocyte-specific Xrcc5 knockout (Xrcc5 cKO) and wildtype (WT) mice that were aged or exposed to DNA damage-inducing chemotherapy were compared. Ovarian follicles and oocytes were quantified, morphologically assessed and analysed via immunohistochemistry for markers of DNA damage and apoptosis. In addition, chemotherapy exposed mice were superovulated, and the numbers and quality of mature metaphase- II (MII) oocytes were assessed. The number of healthy follicles, atretic (dying) follicles, and corpora lutea were similar in Xrcc5 cKO and WT mice at PN50, PN200 and PN300. Additionally, primordial follicle number and ovulation rates were similar in young adult Xrcc5 cKO and WT mice following treatment with cyclophosphamide (75mg/kg), cisplatin (4mg/kg), or vehicle control (saline). Furthermore, KU80 was not essential for the repair of exogenously induced DNA damage in primordial follicle oocytes. These data indicate that KU80 is not required for maintenance of the ovarian reserve, follicle development, or ovulation during maternal ageing. Similarly, this study also indicates that KU80 is not required for the repair of exogenously induced DSBs in the prophase-arrested oocytes of primordial follicles.

XPO1 Enables Adaptive Regulation of mRNA Export Required for Genotoxic Stress Tolerance in Cancer Cells.

XPO1 regulates the dynamic ribonucleoprotein nuclear export in response to genotoxic stress to support tolerance and can be targeted to enhance the sensitivity of cancer cells to endogenous and exogenous DNA damage. See related commentary by Knittel and Reinhardt, p. 3.

Oxidative stress drives mutagenesis through transcription-coupled repair in bacteria

In bacteria, mutations lead to the evolution of antibiotic resistance, which is one of the main public health problems of the twenty-first century. Therefore, determining which cellular processes most frequently contribute to mutagenesis, especially in cells that have not been exposed to exogenous DNA damage, is critical. Here, we show that endogenous oxidative stress is a key driver of mutagenesis and the subsequent development of antibiotic resistance. This is the case for all classes of antibiotics and highly divergent species tested, including patient-derived strains. We show that the transcription-coupled repair pathway, which uses the nucleotide excision repair proteins (TC-NER), is responsible for endogenous oxidative stress-dependent mutagenesis and subsequent evolution. This suggests that a majority of mutations arise through transcription-associated processes rather than the replication fork. In addition to determining that the NER proteins play a critical role in mutagenesis and evolution, we also identify the DNA polymerases responsible for this process. Our data strongly suggest that cooperation between three different mutagenic DNA polymerases, likely at the last step of TC-NER, is responsible for mutagenesis and evolution. Overall, our work identifies a highly conserved pathway that drives mutagenesis due to endogenous oxidative stress, which has broad implications for all diseases of evolution, including antibiotic resistance development.

PRPF19 modulates morphology and growth behavior in a cell culture model of human skin.

The skin provides one of the most visual aging transformations in humans, and premature aging as a consequence of oxidative stress and DNA damage is a frequently seen effect. Cells of the human skin are continuously exposed to endogenous and exogenous DNA damaging factors, which can cause DNA damage in all phases of the cell cycle. Increased levels of DNA damage and/or defective DNA repair can, therefore, accelerate the aging process and/or lead to age-related diseases like cancer. It is not yet clear if enhanced activity of DNA repair factors could increase the life or health span of human skin cells. In previous studies, we identified and characterized the human senescence evasion factor (SNEV)/pre-mRNA-processing factor (PRPF) 19 as a multitalented protein involved in mRNA splicing, DNA repair pathways and lifespan regulation. Here, we show that overexpression of PRPF19 in human dermal fibroblasts leads to a morphological change, reminiscent of juvenile, papillary fibroblasts, despite simultaneous expression of senescence markers. Moreover, conditioned media of this subpopulation showed a positive effect on keratinocyte repopulation of wounded areas. Taken together, these findings indicate that PRPF19 promotes cell viability and slows down the aging process in human skin.

Clamp Loader Processing Is Important during DNA Replication Stress.

The DNA clamp loader is critical to the processivity of the DNA polymerase and coordinating synthesis on the leading and lagging strands. In bacteria, the major subunit of the clamp loader, DnaX, has two forms: the essential full-length τ form and shorter γ form. These are conserved across bacterial species, and three distinct mechanisms have been found to create them: ribosomal frameshift, transcriptional slippage, and, in Caulobacter crescentus, partial proteolysis. This conservation suggests that DnaX processing is evolutionarily important, but its role remains unknown. Here we find a bias against switching from expression of a wild-type dnaX to a nonprocessable τ-only allele in Caulobacter. Despite this bias, cells are able to adapt to the τ-only allele with little effect on growth or morphology and only minor defects during DNA damage. Motivated by transposon sequencing, we find that loss of the gene sidA in the τ-only strain slows growth and increases filamentation. Even in the absence of exogenous DNA damage treatment, the ΔsidA τ-only double mutant shows induction of and dependence on recA, likely due to a defect in resolution of DNA damage or replication fork stalling. We find that some of the phenotypes of the ΔsidA τ-only mutant can be complemented by expression of γ but that an overabundance of τ-only dnaX is also detrimental. The data presented here suggest that DnaX processing is important during resolution of DNA damage events during DNA replication stress. Although the presence of DnaX τ and γ forms is conserved across bacteria, different species have developed different mechanisms to make these forms. This conservation and independent evolution of mechanisms suggest that having two forms of DnaX is important. Despite having been discovered more than 30 years ago, the purpose of expressing both τ and γ is still unclear. Here, we present evidence that expressing two forms of DnaX and controlling the abundance and/or ratio of the forms are important during the resolution of DNA replication stress. IMPORTANCE Though the presence of DnaX τ and γ forms is conserved across bacteria, different species have developed different mechanisms to make these forms. This conservation and independent evolution of mechanisms suggest that having two forms of DnaX is important. Despite having been discovered more than 30 years ago, the purpose of expressing both τ and γ is still unclear. Here, we present evidence that expressing two forms of DnaX and controlling the abundance and/or ratio of the forms is important during the resolution of DNA replication stress.

Circle Damage Sequencing for Whole-Genome Analysis of DNA Damage.

There are many sources of endogenous and exogenous DNA damage. Damaged bases represent a threat to genome integrity and may interfere with normal cellular processes such as replication and transcription. To understand the specificity and biological consequences of DNA damage, it is essential to employ methods that are sensitive enough to detect damaged DNA bases at the level of single nucleotide resolution and genome-wide. Here we describe in detail a method we developed for this purpose, circledamage sequencing (CD-seq). This method is based on the circularization of genomic DNA that contains damaged bases and conversion of the damaged sites into double-strand breaks using specific DNA repair enzymes. Library sequencing of the opened circles yields the precise positions of the DNA lesions that are present. CD-seq can be adopted to various types of DNA damage as long as a specific cleavage scheme can be designed.

Defective DNA polymerase beta invoke a cytosolic DNA mediated inflammatory response.

Base excision repair (BER) has evolved to maintain the genomic integrity of DNA following endogenous and exogenous agent induced DNA base damage. In contrast, aberrant BER induces genomic instability, promotes malignant transformation and can even trigger cancer development. Previously, we have shown that deoxyribo-5'-phosphate (dRP) lyase deficient DNA polymerase beta (POLB) causes replication associated genomic instability and sensitivity to both endogenous and exogenous DNA damaging agents. Specifically, it has been established that this loss of dRP lyase function promotes inflammation associated gastric cancer. However, the way that aberrant POLB impacts the immune signaling and inflammatory responses is still unknown. Here we show that a dRP lyase deficient variant of POLB (Leu22Pro, or L22P) increases mitotic dysfunction associated genomic instability, which eventually leads to a cytosolic DNA mediated inflammatory response. Furthermore, poly(ADP-ribose) polymerase 1 inhibition exacerbates chromosomal instability and enhances the cytosolic DNA mediated inflammatory response. Our results suggest that POLB plays a significant role in modulating inflammatory signaling, and they provide a mechanistic basis for future potential cancer immunotherapies.

An Enzyme-Linked Immunosorbent Assay to Quantify Poly (ADP-Ribose) Level In Vivo.

PolyADP-ribosylation is a posttranslational modification of proteins that results from enzymatic synthesis of poly(ADP-ribose) with NAD+ as the substrate. A unique characteristic of polyADP-ribosylation is that the poly(ADP-ribose) chain can have 200 or more ADP-ribose residues in branched patterns, and the presence and variety of these chains can have substantive effects on protein function. To understand how polyADP-ribosylation affects biological processes, it is important to know the physiological level of poly(ADP-ribose) in cells. Under normal cell physiological conditions and in the absence of any exogenous DNA damaging agents, we found that the concentration of poly(ADP-ribose) in HeLa cells is approximately 0.04pmol (25pg)/106 cells, as measured with a double-antibody sandwich, enzyme-linked immunosorbent assay protocol that avoids artificial activation of PARP1 during cell lysis. Notably, this system demonstrated that the poly(ADP-ribose) level peaks in S phase and that the average cellular turnover of a single poly(ADP-ribose) is less than 40s.

Partial Reduction in BRCA1 Gene Dose Modulates DNA Replication Stress Level and Thereby Contributes to Sensitivity or Resistance.

BRCA1 is a well-known breast cancer risk gene, involved in DNA damage repair via homologous recombination (HR) and replication fork protection. Therapy resistance was linked to loss and amplification of the BRCA1 gene causing inferior survival of breast cancer patients. Most studies have focused on the analysis of complete loss or mutations in functional domains of BRCA1. How mutations in non-functional domains contribute to resistance mechanisms remains elusive and was the focus of this study. Therefore, clones of the breast cancer cell line MCF7 with indels in BRCA1 exon 9 and 14 were generated using CRISPR/Cas9. Clones with successful introduced BRCA1 mutations were evaluated regarding their capacity to perform HR, how they handle DNA replication stress (RS), and the consequences on the sensitivity to MMC, PARP1 inhibition, and ionizing radiation. Unexpectedly, BRCA1 mutations resulted in both increased sensitivity and resistance to exogenous DNA damage, despite a reduction of HR capacity in all clones. Resistance was associated with improved DNA double-strand break repair and reduction in replication stress (RS). Lower RS was accompanied by increased activation and interaction of proteins essential for the S phase-specific DNA damage response consisting of HR proteins, FANCD2, and CHK1.

Targeting DNA damage response as a potential therapeutic strategy for head and neck squamous cell carcinoma.

Cells experience both endogenous and exogenous DNA damage daily. To maintain genome integrity and suppress tumorigenesis, individuals have evolutionarily acquired a series of repair functions, termed DNA damage response (DDR), to repair DNA damage and ensure the accurate transmission of genetic information. Defects in DNA damage repair pathways may lead to various diseases, including tumors. Accumulating evidence suggests that alterations in DDR-related genes, such as somatic or germline mutations, single nucleotide polymorphisms (SNPs), and promoter methylation, are closely related to the occurrence, development, and treatment of head and neck squamous cell carcinoma (HNSCC). Despite recent advances in surgery combined with radiotherapy, chemotherapy, or immunotherapy, there has been no substantial improvement in the survival rate of patients with HNSCC. Therefore, targeting DNA repair pathways may be a promising treatment for HNSCC. In this review, we summarized the sources of DNA damage and DNA damage repair pathways. Further, the role of DNA damage repair pathways in the development of HNSCC and the application of small molecule inhibitors targeting these pathways in the treatment of HNSCC were focused.

The SKP2-p27 axis defines susceptibility to cell death upon CHK1 inhibition.

Checkpoint kinase 1 (CHK1; encoded by CHEK1) is an essential gene that monitors DNA replication fidelity and prevents mitotic entry in the presence of under‐replicated DNA or exogenous DNA damage. Cancer cells deficient in p53 tumor suppressor function reportedly develop a strong dependency on CHK1 for proper cell cycle progression and maintenance of genome integrity, sparking interest in developing kinase inhibitors. Pharmacological inhibition of CHK1 triggers B‐Cell CLL/Lymphoma 2 (BCL2)‐regulated cell death in malignant cells largely independently of p53, and has been suggested to kill p53‐deficient cancer cells even more effectively. Next to p53 status, our knowledge about factors predicting cancer cell responsiveness to CHK1 inhibitors is limited. Here, we conducted a genome‐wide CRISPR/Cas9‐based loss‐of‐function screen to identify genes defining sensitivity to chemical CHK1 inhibitors. Next to the proapoptotic BCL2 family member, BCL2 Binding Component 3 (BBC3; also known as PUMA), the F‐box protein S‐phase Kinase‐Associated Protein 2 (SKP2) was validated to tune the cellular response to CHK1 inhibition. SKP2 is best known for degradation of the Cyclin‐dependent Kinase Inhibitor 1B (CDKN1B; also known as p27), thereby promoting G1‐S transition and cell cycle progression in response to mitogens. Loss of SKP2 resulted in the predicted increase in p27 protein levels, coinciding with reduced DNA damage upon CHK1‐inhibitor treatment and reduced cell death in S‐phase. Conversely, overexpression of SKP2, which consequently results in reduced p27 protein levels, enhanced cell death susceptibility to CHK1 inhibition. We propose that assessing SKP2 and p27 expression levels in human malignancies will help to predict the responsiveness to CHK1‐inhibitor treatment.